Multiple functional neurosteroid binding sites on GABAA receptors.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1ß3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and ß3 (labeled residue ß3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ß3-L294 and G308) in the interface between the ß3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and ß3-α1 intersubunit sites are critical for neurosteroid action.

Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate α1ß2γ2L GABAA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone.

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABAA receptor. The sites are located in the membrane-spanning domains of the receptor at the ß-α subunit interface (site I) and within the α (site II) and ß subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1ß2γ2L GABAA receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.

Mapping nephron mass in vivo using positron emission tomography.

Nephron number varies widely in humans. A low nephron endowment at birth or a loss of functioning nephrons is strongly linked to increased susceptibility to chronic kidney disease. In this work, we developed a contrast agent, radiolabeled cationic ferritin (RadioCF), to map functioning glomeruli in vivo in the kidney using positron emission tomography (PET). PET radiotracers can be detected in trace doses (<30 nmol), making them useful for rapid clinical translation. RadioCF is formed from cationic ferritin (CF) and with a radioisotope, Cu-64, incorporated into the ferritin core. We showed that RadioCF binds specifically to kidney glomeruli after intravenous injection in mice, whereas radiolabeled noncationic ferritin (RadioNF) and free Cu-64 do not. We then showed that RadioCF-PET can distinguish kidneys in healthy wild-type (WT) mice from kidneys in mice with oligosyndactylism (Os/+), a model of congenital hypoplasia and low nephron mass. The average standardized uptake value (SUV) measured by PET 90 min after injection was 21% higher in WT mice than in Os/+ mice, consistent with the higher glomerular density in WT mice. The difference in peak SUV from SUV at 90 min correlated with glomerular density in male mice from both WT and Os/+ cohorts (R2 = 0.98). Finally, we used RadioCF-PET to map functioning glomeruli in a donated human kidney. SUV within the kidney correlated with glomerular number (R2= 0.78) measured by CF-enhanced magnetic resonance imaging in the same locations. This work suggests that RadioCF-PET appears to accurately detect nephron mass and has the potential for clinical translation.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

"Click Chip" Conjugation of Bifunctional Chelators to Biomolecules.

There is a growing demand for diagnostic procedures including in vivo tumor imaging. Radiometal-based imaging agents are advantageous for tumor imaging because radiometals (i) have a wide range of half-lives and (ii) are easily incorporated into imaging probes via a mild, rapid chelation event with a bifunctional chelator (BFC). Microfluidic platforms hold promise for synthesis of radiotracers because they can easily handle minute volumes, reduce consumption of expensive reagents, and minimize personnel exposure to radioactive compounds. Here we demonstrate the use of a "click chip" with an immobilized Cu(I) catalyst to facilitate the "click chemistry" conjugation of BFCs to biomolecules (BMs); a key step in the synthesis of radiometal-based imaging probes. The "click chip" was used to synthesize three different BM-BFC conjugates with minimal amounts of copper present in reaction solutions (∼20 ppm), which reduces or obviates the need for a copper removal step. These initial results are promising for future endeavors of synthesizing radiometal-based imaging agents completely on chip.

Synthesis, pharmacological evaluation and molecular modeling studies of triazole containing dopamine D3 receptor ligands.

A series of 2-methoxyphenyl piperazine analogues containing a triazole ring were synthesized and their in vitro binding affinities at human dopamine D2 and D3 receptors were evaluated. Compounds 5b, 5c, 5d, and 4g, demonstrate high affinity for dopamine D3 receptors and moderate selectivity for the dopamine D3 versus D2 receptor subtypes. To further examine their potential as therapeutic agents, their intrinsic efficacy at both D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay. Affinity at dopamine D4 and serotonin 5-HT1A receptors was also determined. In addition, information from previous molecular modeling studies of the binding of a panel of 163 structurally-related benzamide analogues at dopamine D2 and D3 receptors was applied to this series of compounds. The results of the modeling studies were consistent with our previous experimental data. More importantly, the modeling study results explained why the replacement of the amide linkage with the hetero-aromatic ring leads to a reduction in the affinity of these compounds at D3 receptors.

Triazine-based tool box for developing peptidic PET imaging probes: syntheses, microfluidic radiolabeling, and structure-activity evaluation.

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvß3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using "click" chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with (64)Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I(125)-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-"Click"-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.

Thiolene and SIFEL-based Microfluidic Platforms for Liquid-Liquid Extraction.

Microfluidic platforms provide several advantages for liquid-liquid extraction (LLE) processes over conventional methods, for example with respect to lower consumption of solvents and enhanced extraction efficiencies due to the inherent shorter diffusional distances. Here, we report the development of polymer-based parallel-flow microfluidic platforms for LLE. To date, parallel-flow microfluidic platforms have predominantly been made out of silicon or glass due to their compatibility with most organic solvents used for LLE. Fabrication of silicon and glass-based LLE platforms typically requires extensive use of photolithography, plasma or laser-based etching, high temperature (anodic) bonding, and/or wet etching with KOH or HF solutions. In contrast, polymeric microfluidic platforms can be fabricated using less involved processes, typically photolithography in combination with replica molding, hot embossing, and/or bonding at much lower temperatures. Here we report the fabrication and testing of microfluidic LLE platforms comprised of thiolene or a perfluoropolyether-based material, SIFEL, where the choice of materials was mainly guided by the need for solvent compatibility and fabrication amenability. Suitable designs for polymer-based LLE platforms that maximize extraction efficiencies within the constraints of the fabrication methods and feasible operational conditions were obtained using analytical modeling. To optimize the performance of the polymer-based LLE platforms, we systematically studied the effect of surface functionalization and of microstructures on the stability of the liquid-liquid interface and on the ability to separate the phases. As demonstrative examples, we report (i) a thiolene-based platform to determine the lipophilicity of caffeine, and (ii) a SIFEL-based platform to extract radioactive copper from an acidic aqueous solution.

Evaluation of N-phenyl homopiperazine analogs as potential dopamine D3 receptor selective ligands.

A series of N-(2-methoxyphenyl)homopiperazine analogs was prepared and their affinities for dopamine D2, D3, and D4 receptors were measured using competitive radioligand binding assays. Several ligands exhibited high binding affinity and selectivity for the D3 dopamine receptor compared to the D2 receptor subtype. Compounds 11a, 11b, 11c, 11f, 11j and 11k had K(i) values ranging from 0.7 to 3.9 nM for the D3 receptor with 30- to 170-fold selectivity for the D3 versus D2 receptor. Calculated logP values (logP=2.6-3.6) are within the desired range for passive transport across the blood-brain barrier. When the binding and the intrinsic efficacy of these phenylhomopiperazines was compared to those of previously published phenylpiperazine analogues, it was found that (a) affinity at D2 and D3 dopamine receptors generally decreased, (b) the D3 receptor binding selectivity (D2:D3 K(i) value ratio) decreased and, (c) the intrinsic efficacy, measured using a forskolin-dependent adenylyl cyclase inhibition assay, generally increased.

Synthesis and Expression of a Targeted, Ferritin-Based Tracer for PET Imaging of Kidney Glomeruli.

Cationic ferritin (CF) has been developed as a multimodal, targeted imaging tracer to directly detect and map nephrons in the kidney in vivo. Direct detection of functional nephrons provides a unique, sensitive biomarker to predict or monitor kidney disease progression. CF has been developed to map functional nephron number from magnetic resonance imaging (MRI) or positron emission tomography (PET). Previous preclinical imaging studies have used non-human-derived ferritin and commercial formulations that must still be developed for translation to clinical use. Here we describe the reproducible formulation of CF (either derived from horse or from human recombinant ferritin) optimized for intravenous injection and radiolabeling by PET. The human recombinant heteropolymer ferritin is spontaneously assembled in liquid culture (Escherichia coli, E. coli) and modified to form human recombinant cationic ferritin (HrCF) to mitigate potential immunologic reactions for use in humans.

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ß3 subunit of the GABA(A) receptor.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5ß)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-ß3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-ß3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 µM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-ß3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ß3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3ß,5ß)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-ß3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ß3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of ß-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-ß-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a ß-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and ß-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Microfluidic labeling of biomolecules with radiometals for use in nuclear medicine.

Radiometal-based radiopharmaceuticals, used as imaging and therapeutic agents in nuclear medicine, consist of a radiometal that is bound to a targeting biomolecule (BM) using a bifunctional chelator (BFC). Conventional, macroscale radiolabeling methods use an excess of the BFC-BM conjugate (ligand) to achieve high radiolabeling yields. Subsequently, to achieve maximal specific activity (minimal amount of unlabeled ligand), extensive chromatographic purification is required to remove unlabeled ligand, often resulting in longer synthesis times and loss of imaging sensitivity due to radioactive decay. Here we describe a microreactor that overcomes the above issues through integration of efficient mixing and heating strategies while working with small volumes of concentrated reagents. As a model reaction, we radiolabel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated to the peptide cyclo(Arg-Gly-Asp-DPhe-Lys) with (64)Cu(2+). We show that the microreactor (made from polydimethylsiloxane and glass) can withstand 260 mCi of activity over 720 hours and retains only minimal amounts of (64)Cu(2+) (<5%) upon repeated use. A direct comparison between the radiolabeling yields obtained using the microreactor and conventional radiolabeling methods shows that improved mixing and heat transfer in the microreactor leads to higher yields for identical reaction conditions. Most importantly, by using small volumes (~10 µL) of concentrated solutions of reagents (>50 µM), yields of over 90% can be achieved in the microreactor when using a 1:1 stoichiometry of radiometal to BFC-BM. These high yields eliminate the need for use of excess amounts of often precious BM and obviate the need for a chromatographic purification process to remove unlabeled ligand. The results reported here demonstrate the potential of microreactor technology to improve the production of patient-tailored doses of radiometal-based radiopharmaceuticals in the clinic.

Subtype selectivity of dopamine receptor ligands: insights from structure and ligand-based methods.

Subtype selective dopamine receptor ligands have long been sought after as therapeutic and/or imaging agents for the treatment and monitoring of neurologic disorders. We report herein on a combined structure- and ligand-based approach to explore the molecular mechanism of the subtype selectivity for a large class of D2-like dopamine receptor ligands (163 ligands in total). Homology models were built for both human D(2L) and D3 receptors in complex with haloperidol. Other ligands, which included multiple examples of substituted phenylpiperazines, were aligned against the binding conformations of haloperidol, and three-dimensional quantitative structure activity relationship (3D-QSAR) analyses were carried out. The receptor models show that although D2 and D3 share highly similar folds and 3D conformations, the slight sequence differences at their extracellular loop regions result in the binding cavity in D2 being comparably shallower than in D3, which may explain why some larger ligands bind with greater affinity at D3 compared to D2 receptors. The QSAR models show excellent correlation and high predictive power even when evaluated by the most stringent criteria. They confirm that the origins of subtype selectivity for the ligands arise primarily due to differences in the contours of the two binding sites. The predictive models suggest that while both steric and electrostatic interactions contribute to the compounds' binding affinity, the major contribution arises from hydrophobic interactions, with hydrogen bonding conferring binding specificity. The current work provides clues for the development of more subtype selective dopamine receptor ligands. Furthermore, it demonstrates the possibility of being able to apply similar modeling methods to other subtypes or classes of receptors to study GPCR receptor-ligand interactions at a molecular level.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1ß3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3ß-epimer epi-allopregnanolone, binds to the canonical ß3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the ß3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

[18F]- and [11C]-labeled N-benzyl-isatin sulfonamide analogues as PET tracers for apoptosis: synthesis, radiolabeling mechanism, and in vivo imaging study of apoptosis in Fas-treated mice using [11C]WC-98.

The radiolabeled isatin sulfonamide caspase-3 inhibitor, [18F] 2 (WC-II-89), is a potential PET radiotracer for noninvasive imaging of apoptosis. The radiolabeling mechanism was studied by 13C NMR, ESI/MS, and computational calculations. It was found that the high electrophilicity of the C3 carbonyl group in the isatin ring, which served as a trap for [18F]fluoride, was responsible for the failure of the radiolabeling via nucleophilic substitution of the mesylate group in 7a by [18F]fluoride. Once treated with a strong base, 7a opened the isatin ring completely to form an isatinate intermediate 16, which lost the ability to trap [18F]fluoride, thereby allowing the displacement of the mesylate group to afford the 18F-labeled isatinate 17. [18F] 17 can be converted to isatin [18F] 2 efficiently under acidic conditions. The ring-opening and re-closure of the isatin ring under basic and acidic conditions were confirmed by reversed phase HPLC analysis, ESI/MS and 13C NMR studies. Computational studies of model compounds also support the above proposed mechanism. Similarly, the ring-opening and re-closure method was used successfully in the synthesis of the 11C labeled isatin sulfonamide analogue [11C] 4 (WC-98). A microPET imaging study using [11C] 4 in the Fas liver apoptosis model demonstrated retained activity in the target organ (liver) of the treated mice. Increased caspase-3 activation in the liver was verified by the fluorometric caspase-3 enzyme assay. Therefore, this study provides a useful method for radio-synthesis of isatin derivative radiotracers for PET and SPECT studies, and [11C] 4 is a potential PET radiotracer for noninvasive imaging of apoptosis.

The molecular determinants of neurosteroid binding in the GABA(A) receptor.

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

Assessment of Copper Nanoclusters for Accurate in Vivo Tumor Imaging and Potential for Translation.

Nanoparticles have been widely used for preclinical cancer imaging. However, their successful clinical translation is largely hampered by potential toxicity, unsatisfactory detection of malignancy at early stages, inaccurate diagnosis of tumor biomarkers, and histology for imaging-guided treatment. Herein, a targeted copper nanocluster (CuNC) is reported with high potential to address these challenges for future translation. Its ultrasmall structure enables efficient renal/bowel clearance, minimized off-target effects in nontargeted organs, and low nonspecific tumor retention. The pH-dependent in vivo dissolution of CuNCs affords minimal toxicity and potentially selective drug delivery to tumors. The intrinsic radiolabeling through the direct addition of 64Cu to CuNC (64Cu-CuNCs-FC131) synthesis offers high specific activity for sensitive and accurate detection of CXCR4 via FC131-directed targeting in novel triple negative breast cancer (TNBC) patient-derived xenograft mouse models and human TNBC tissues. In summary, this study not only reveals the potential of CXCR4-targeted 64Cu-CuNCs for TNBC imaging in clinical settings, but also provides a useful strategy to design and assess the translational potential of nanoparticles for cancer theranostics.

Mutations of the GABA-A receptor alpha1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues alpha1Asn407/Tyr410 in the M4 transmembrane domain and residue alpha1Gln241 in the M1 domain. We examined the role of residues in the alpha1 subunit M1 domain in the modulation of the rat alpha1beta2gamma2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the alpha1Q241W or alpha1Q241L mutations were insensitive to (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP), albeit with different underlying mechanisms. The alpha1Q241S mutant was potentiated by 3alpha5alphaP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the alpha1Q241L mutation had no effect on channel potentiation by (3alpha,5alpha)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, alpha1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5alpha-pregnan-20-one) potentiated the wild-type receptor but not the alpha1Q241L mutant. The findings are consistent with a model in which the alpha1Ser240 and alpha1Gln241 residues shape the surface to which steroid molecules bind.